Briefly, and as described in more detail below, described herein is a method of depositing a decellularized extracellular matrix, e.g., a mesenchymal stem cell-secreted extracellular matrix, on biomaterials, e.g., polymeric implants of any size and geometry. This matrix is produced by cells, for example, MSCs derived from any tissue compartment (e.g., bone marrow, adipose tissue, muscle, dental pulp, etc.) on, e.g., tissue culture plastic (TCP) under controlled conditions. The result is then decellularized and removed from the TCP, resulting in a composition of the cell-secreted components without the antigenic cellular structures or contaminating DNA. The application and implantation of this decellularized extracellular matrix to supports accelerates tissue formation in a natural manner and has the opportunity to make implantable materials more patient-friendly and enhance integration into the patient by presenting a cell-derived surface coating.
Several features of the current approach should be noted. Unlike the coating of implants with homogeneous proteins (e.g., collagen I, fibronectin, vitronectin) that have previously demonstrated increased bone formation, the deposition of the complex array of proteins and polysaccharides included in the endogeneous cell-secreted ECM provides cells with a natural substrate for interaction, thereby enhancing cellular adhesion, viability, survival, and tissue formation. Moreover, the matrix can be generated from patients or an unrelated donor without concerns of immunogenicity due to the nature of MSCs and DNase treatment of these matrices, thereby addressing the concerns of immune response for ˜4% of the population with allergies to collagen.
Disclosed herein are methods for producing compositions of decellularized extracellular matrix via tissue culture and use of the compositions for coating biomaterials such as tissue culture substrates, osteogenic gels, and medical devices.
Disclosed herein is a method for producing a composition comprising a decellularized extracellular matrix (DM), comprising a) obtaining a population of cells grown on a tissue culture substrate under conditions sufficient to form an extracellular matrix (ECM); b) removing the cells from the tissue culture substrate to form a tissue culture substrate coated with DM; c) separating the DM from the tissue culture substrate into a solvent to form a solution comprising the DM; and d) dissociating the DM in the solution, thereby producing the composition comprising the DM.
In some aspects, step a) comprises growing a population of human mesenchymal stem cells (MSCs) derived from bone marrow on a tissue culture substrate comprising tissue culture plastic (TCP) by seeding MSCs at 50,000 cells/cm2 and maintaining the MSCs in ambient oxygen at 21% O2 in alpha modified Eagle's medium (MEM) supplemented with 50 μg/ml ascorbate-2-phosphate for 15 days to form an ECM; wherein step b) comprises removing the cells from the tissue culture substrate by treatment with 0.5% Triton X-100 in 20 mM ammonium hydroxide (NH4OH) in phosphate buffered saline (PBS) for 5 minutes at 37 degrees C. to form a tissue culture substrate coated with DM; wherein step c) comprises separating the DM from the tissue culture substrate by treatment with 0.02 N acetic acid and scraping the DM from the tissue culture substrate into the 0.02 N acetic acid to form a solution comprising the DM; and wherein step d) comprises dissociating the DM in the solution by sonication, thereby producing the composition comprising the DM.
In some aspects, the methods further include transferring the dissociated DM to a gelatinous support. In some aspects, the methods further include treating the tissue culture substrate coated with DM with a nuclease. In some aspects, the methods further include treating the tissue culture substrate coated with DM with a DNase. In some aspects, the methods further include drying the tissue culture substrate coated with DM. In some aspects, the methods further include lyophilizing the composition comprising the DM. In some aspects, the methods further include transferring the dissociated DM to a solid support.
In some aspects, the ECM is osteogenic, chondrogenic, myogenic, adipogenic, keratinogenic, keratogenic, neurogenic, tenogenic, angiogenic, urotheliogenic, hepatogenic, or nephrogenic. In some aspects, the ECM is osteogenic. In some aspects, the DM is effective at maintaining stem and progenitor cells in an undifferentiated or minimally differentiated state for the purpose of expansion and cell study.
In some aspects, the cells are mesenchymal stem cells (MSCs). In some aspects, the MSC are derived from bone marrow, adipose tissue, muscle, periodontal tissue, or dental pulp. In some aspects, the MSC are human, mouse, rat, dog, cat, rabbit, horse, pig, or nonhuman primate. In some aspects, the cells are human MSC. In some aspects, the cells are human MSC derived from bone marrow. In some aspects, the cells are obtained in a culture medium. In some aspects, the cells are obtained in Dulbecco's Modified Eagle's Medium.
In some aspects, the tissue culture substrate is tissue culture plastic (TCP) or glass or a bioceramic or natural proteins and polymers such as collagen or fibrin or substrates derived from synthetic polymers such as polycaprolactone or poly(lactide-co-glycolide) and its homopolymers or thermoresponsive materials such as poly(N-isopropylacrylamide). In some aspects, the tissue culture substrate is TCP. In some aspects, the conditions sufficient to form an ECM comprise seeding human MSCs at high density greater than or equal to 50,000 cells/sq. cm and maintaining in ambient oxygen at 21% O2 in alpha modified Eagle's medium supplemented with 50-100 μg/ml ascorbate-2-phosphate for 15 days.
In some aspects, removing the cells from the tissue culture substrate is performed by treatment with detergent or by freeze/thaw cycles. In some aspects, removing the cells from the tissue culture substrate is performed by treatment with 0.5% Triton X-100 in 20 mM NH4OH in phosphate buffered saline (PBS) for 5 minutes at 37 degrees C.
In some aspects, separating the DM from the tissue culture substrate comprises treatment with an acidic solvent and mechanical removal of the DM from the tissue culture substrate or scraping or lifting the DM from thermoresponsive polymers by reducing the temperature. In some aspects, separating the DM from the tissue culture substrate comprises treatment with an acidic solvent and scraping the DM from the tissue culture substrate. In some aspects, the solvent comprises 0.02 N acetic acid.
In some aspects, dissociating the DM comprises sonication or mechanical homogenization or enzyme treatment. In some aspects, dissociating the DM comprises sonication.
Also described herein is a method for producing a composition comprising a decellularized osteogenic extracellular matrix (oDM) produced in tissue culture, the method comprising a) growing a population of human MSC derived from bone marrow on a tissue culture substrate comprising TCP by seeding human MSCs at high density (greater than or equal to 50,000 cells/sq. cm) and maintaining in ambient oxygen (21% O2) in alpha modified Eagle's medium supplemented with 50 μg/ml ascorbate-2-phosphate for 15 days to form an extracellular matrix (ECM); b) removing the cells from the tissue culture substrate by treatment with 0.5% Triton X-100 in 20 mM NH4OH in PBS for 5 minutes at 37 degrees C. to form tissue culture substrate coated with oDM; c) separating the oDM from the tissue culture substrate by treatment with 0.02 N acetic acid and scraping the oDM from the tissue culture substrate into the 0.02 N acetic acid to form a solution comprising the oDM; and d) dissociating the oDM in the solution by sonication, thereby producing the composition comprising the osteogenic decellularized ECM.
Also described herein is a method for producing a composition comprising a decellularized extracellular matrix (DM) adsorbed with a biologically active material (BAM), comprising a) obtaining a population of cells grown on a tissue culture substrate under conditions sufficient to form an extracellular matrix (ECM); b) removing the cells from the tissue culture substrate to form a tissue culture substrate coated with DM; c) contacting the DM with the BAM under conditions sufficient for adsorption of the BAM by the DM; d) separating the DM from the tissue culture substrate into a solvent to form a solution comprising the DM adsorbed with BAM; and d) dissociating the DM in the solution, thereby producing the composition comprising the DM adsorbed with BAM. In some aspects, the biologically active material is a therapeutic agent, a small molecule, a nucleic acid, or a protein molecule. In some aspects, the biologically active material is a protein molecule that is differentially expressed in diabetes patients. Also described herein is a support contacted with the BAM contacted DM produced by any of the methods described herein. In some aspects, the support is an alginate gel.
Also described herein is a method of producing a composition comprising a decellularized extracellular matrix (DM) produced in tissue culture, comprising a) obtaining a population of cells on a thermoresponsive polymer at 37° C. under conditions sufficient to form an extracellular matrix (ECM); b) removing the cells with a hypotonic solution to form the composition comprising the DM; and c) reducing the temperature from 37° C. to 20° C. In some aspects, the thermoresponsive polymer is Poly(N-isopropylacrylamide) (PIPAAm). In some aspects, the thermoresponsive polymer is described in U.S. Pat. Nos. 6,451,429; 6,410,644; 6,410,155; 6,277,768; and 5,969,052; each of which is herein incorporated by reference in its entirety for all purposes.
Also described herein is a method for producing a composition comprising a decellularized extracellular matrix (DM) produced in tissue culture, comprising a) obtaining a population of human MSCs on a tissue culture substrate under conditions sufficient to form an extracellular matrix (ECM); and b) removing the cells from the tissue culture substrate with a hypotonic solution to form the composition comprising the DM. In some aspects, step a) comprises growing a population of human MSC derived from bone marrow on a tissue culture substrate comprising tissue culture plastic (TCP) by seeding human MSCs at 50,000 cells/cm2 and maintaining the MSCs in ambient oxygen at 21% O2 in alpha modified Eagle's medium (MEM) supplemented with 50 μg/ml ascorbate-2-phosphate for 15 days to form an extracellular matrix (ECM); and wherein step b) comprises removing the cells from the tissue culture substrate by treatment with 0.5% Triton X-100 in 20 mM NH4OH in PBS for 5 minutes at 37 degrees C. to form the composition comprising the DM.
Also described herein is a method for producing an osteogenic extracellular matrix comprising obtaining a population of human MSC seeded on a tissue culture substrate and maintaining the MSCs in ambient oxygen in a medium supplemented with ascorbate-2-phosphate under conditions sufficient to produce the osteogenic extracellular matrix.
Also described herein is a method for producing a support coated with a DM comprising contacting a support with a composition comprising a decellularized ECM produced by a method described herein.
In some aspects, the support is a second tissue culture substrate comprising TCP. In some aspects, the support comprises a microsphere. In some aspects, the support is a three dimensional polymeric biomaterial. In some aspects, the support is an alginate hydrogel. In some aspects, the support is a medical device. In some aspects, the support comprises metal.
Also described herein is a DM coated support produced by a method described herein.
Also described herein is a method for producing a support coated with a DM comprising contacting a support with a solution comprising a decellularized ECM (DM) derived from human MSCs. In some aspects, the DM is solubilized and homogenous in the solution.
Also described herein is a composition comprising a decellularized ECM produced by a method described herein. In some aspects, the composition further comprises a support, wherein the support is a bioceramic.
Also described herein is a solution comprising a solubilized and homogenously distributed decellularized ECM (DM) derived from human MSCs.
Also described herein is a composition comprising a decellularized ECM (DM) derived from human MSCs, wherein the DM has a protein content of about 10-20 μg/cm2, wherein the DM has 99% less DNA as compared to a non-decellularized ECM control sample, and wherein the DM expresses type 1 collagen, fibronectin, biglycan, and, decorin. In some aspects, the composition further comprises a solid or gelatinous support.
Also described herein is a composition comprising human MSCs contacted with a decellularized ECM (DM) derived from human MSCs. In some aspects, the composition further comprises a solid or gelatinous support.
Also described herein is a composition comprising a solubilized, decellularized ECM, wherein the ECM is produced by tissue culture cells. In some aspects, the tissue culture cells are human cells. In some aspects, the composition further comprises a support, wherein the support is a bioceramic.
Also described herein is a support coated with a composition described herein.
Also described herein is a composition comprising a decellularized ECM produced by a method described herein and a crosslinking agent. In some aspects, the crosslinking agent is 1-Ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride (EDC).